Purification and characterization of β -agarase from agar-liquefying soi bacterium Acinetobacter sp., AG LSL-1

Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka, India
PROCESS BIOCHEMISTRY (Impact Factor: 2.52). 09/2009; 44(9):999-1003. DOI: 10.1016/j.procbio.2009.04.025


The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg−1 protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg2+, Ag+ and Cu2+, whereas 25–40% of native activity was retained in the presence of Zn2+, Sn2+ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species.

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    • "SY37-12 (Wang et al. 2006), Agarivorans albus YKW-34 (Fu et al. 2008), and Acinetobacter sp. Ag LSL-1 (Lakshmikanth et al. 2009) retained activities after incubation as follows; 20 % after 1 min at 70 °C, 10 % after 60 min at 70 °C, and none after 60 min at 60 °C, respectively. "
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    ABSTRACT: An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl2 or MgCl2. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl2, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea. Electronic supplementary material The online version of this article (doi:10.1007/s00792-013-0575-z) contains supplementary material, which is available to authorized users.
    Extremophiles 08/2013; 17(6). DOI:10.1007/s00792-013-0575-z · 2.31 Impact Factor
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    • "It is remarkable that the agarase from Acinetobacter sp. AGLSL-1 show a specific activity of 397 U/mg [31], which is the highest among the native agarases have been reported up to date. There are no special properties as comparing the above four agarases with marine derived agarases in pH and temperature properties. "
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    Marine Drugs 01/2010; 8(1):200-18. DOI:10.3390/md8010200 · 2.85 Impact Factor
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    Biotechnology and Bioprocess Engineering 06/2011; 16(3):513-519. DOI:10.1007/s12257-010-0399-y · 1.11 Impact Factor
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